www.elsevier.com/locate/ymcne

Mol. Cell. Neurosci. 27 (2004) 441–452

HGF promotes survival and growth of maturing sympathetic neurons

by PI-3 kinase- and MAP kinase-dependent mechanisms

Jane Thompson,a,b,* Xavier Dolcet,a,c Mark Hilton,a Mary Tolcos,a,d and Alun M. Daviesa,e

aDepartment of Preclinical Veterinary Sciences, Royal (Dick) School of Veterinary Studies, University of Edinburgh, Edinburgh EH9 1QH, Scotland,

United KingdombFujisawa Institute of Neuroscience, Edinburgh EH8 9JE, Scotland, United KingdomcLab Anatomia Patologica-Genetica, Hospital Arnau de Vilanova, 25198 Lleida, SpaindDepartment of Anatomy and Cell Biology, University of Melbourne, Parkville, VIC, 3010 AustraliaeSchool of Biosciences, Cardiff CF10 3US, Wales, United Kingdom

Received 26 April 2004; revised 26 July 2004; accepted 27 July 2004

Available online 28 October 2004

Hepatocyte growth factor (HGF) is a pleiotrophic factor whose many

functions include promoting neuronal survival and growth. Hitherto,

these effects have been observed in the presence of other neurotrophic

factors like NGF and CNTF, and this requirement for an accessory

factor has made it difficult to elucidate the signaling pathways that

mediate its survival and growth-enhancing effects. Here, we show that

HGF promotes the survival of mature sympathetic neurons of the

superior cervical ganglion (SCG) grown at low density in defined

medium lacking other neurotrophic factors. This effect was first clearly

observed in cultures established from postnatal day 20 (P20) mice and

became maximal by P40. HGF also enhanced the growth of neurite

arbors from neurons throughout postnatal development and in the

adult. HGF treatment resulted in phosphorylation of Akt and ERK1/

ERK2. Preventing Akt activation with the phosphatidylinositol-3 (PI-3)

kinase inhibitor LY294002 blocked the HGF survival response, and

inhibition of ERK activation with the MEK inhibitors PD98059 or

U0126 reduced the HGF survival response and the neurite growth-

promoting effects of HGF. These results indicate that HGF promotes

the survival and growth of maturing sympathetic neurons by both PI-3

kinase- and MAP kinase-dependent mechanisms.

D 2004 Elsevier Inc. All rights reserved.

Introduction

Hepatocyte growth factor (HGF), also known as Scatter factor,

is a mutlifunctional cytokine that exerts a variety of effects on

many cell types. It was first identified as a potent mitogen for

hepatocytes (Montesano et al., 1991; Nakamura et al., 1989) and

1044-7431/$ - see front matter D 2004 Elsevier Inc. All rights reserved.

doi:10.1016/j.mcn.2004.07.007

* Corresponding author. Fujisawa Institute of Neuroscience in Edin-

burgh, Level 6, Appleton Tower, Crichton Street, Edinburgh, EH8 9JE,

Scotland, United Kingdom. Fax: +44 131 667 9381.

E-mail address: Jane.Thompson@ed.ac.uk (J. Thompson).

Available online on ScienceDirect (www.sciencedirect.com.)

has subsequently been shown to play a role in morphogenesis,

angiogenesis, and tumorogenesis. The effects of HGF are mediated

via the receptor tyrosine kinase Met. (Bladt et al., 1995; Tsarfaty et

al., 1992). Gene-targeting experiments in mice have shown that

HGF and Met are required for the development of the placenta,

liver, and skeletal muscle of the limbs and trunk (Bladt et al., 1995;

Maina et al., 1996; Schmidt et al., 1995; Uehara et al., 1995).HGF

promotes the survival and proliferation of several cell types,

including Schwann cells, chondrocytes, keratinocytes, and leio-

myosarcoma cells, and stimulates the migration and dissociation of

epithelial sheets (Brinkmann et al., 1995; Gheradi and Stoker,

1991; Krasnoselsky et al., 1994; Mildner et al., 2002; Takebayashi

et al., 1995; Xiao et al., 2000).

HGF plays a role in several aspects of neural development

(Maina and Klein, 1999). Experiments in chick embryos have

suggested that HGF might play a role neural induction (Bronner-

Fraser, 1995). Later in embryonic development and in the adult

nervous system, a variety of neurons and glial cells express HGF

and Met (Andermarcher et al., 1996; Di Renzo et al., 1993; Jung et

al., 1994; Krasnoselsky et al., 1994; Maina et al., 1997, 1998;

Sonnenberg et al., 1993; Thewke and Seeds, 1996, 1999). HGF

promotes the survival of a subset of motoneurons and has been

implicated in guiding motor axons to their targets (Caton et al.,

2000; Ebens et al., 1996; Novak et al., 2000; Wong et al., 1997;

Yamamoto et al., 1997). HGF cooperates with CNTF to promote

motoneuron and parasympathetic neuron survival (Davey et al.,

2000; Wong et al., 1997) and with NGF to promote sensory neuron

survival and outgrowth of sensory and sympathetic axons (Maina

et al., 1997, 1998; Yang et al., 1998). HGF enhances the number of

tyrosine hydroxylase-positive neurons in midbrain cultures (Hama-

noue et al., 1996), protects cerebellar granule neurons from

apoptosis following serum withdrawal (Zhang et al., 2000), and

promotes neurite growth from a subset of hippocampal neurons in

culture (Korhonen et al., 2000). HGF also rescues hippocampal

CA1 neurons following transient global ischemia (Miyazawa et al.,

J. Thompson et al. / Mol. Cell. Neurosci. 27 (2004) 441–452442

1998) and cerebellar granule neurons following NMDA excitotoxi-

city (Hossain et al., 2002).

Binding of HGF to Met activates cytoplasmic effectors via

multifunctional docking sites located in its intracellular domain

(Ponzetto et al., 1994). In nonneuronal cells, Met can activate

several signaling cascades, including the Ras/MAP kinase and

JNK/SAP kinase pathways via Grb2 and Gab1, phospholipid

pathways through binding of phosphatidylinositol-3 kinase (PI3K)

and phospholipase C (PLC), phosphotyrosine-mediated pathways

through interaction with Src tyrosine kinase, SHP2 tyrosine

phosphatase, Nck, and the STAT pathway (Boccaccio et al.,

1998; Kochhar and Iyer, 1996; Nguyen et al., 1997; Ponzetto et al.,

1994; Rodrigues et al., 1997; Weidner et al., 1995). Studies of mice

that express Met receptors in which the multifunctional docking

sites have been replaced with specific binding motifs for PI3K, Src,

or Grb2 have shown that signaling through PI3K is sufficient to

promote outgrowth of specific motor axons in vivo and to mediate

the growth-promoting effects of HGF on sympathetic neurites in

vitro (Maina et al., 2001). However, the signaling pathways that are

important for neuronal survival have not been ascertained.

Previous work has shown that HGF enhances the survival and

differentiation of sympathetic neuroblasts via an autocrine mech-

anism and subsequently increases the growth of neurites from

postmitotic embryonic sympathetic neurons grown in the presence

of NGF but does not enhance the survival of these neurons either

alone or in the presence of NGF (Maina et al., 1998). Here, we

have investigated effects of HGF on postnatal and adult sympa-

thetic neurons and found that it promotes their survival and growth

in culture in the absence of other neurotrophic factors. This finding

has in turn permitted us to investigate the signaling pathways that

are important for survival and have found that PI3K/Akt and, to a

lesser extent, MAP kinase signaling mediate the HGF survival

response.

Fig. 1. Bar charts of the numbers of P1, P10, P20, and P40 SCG neurons

surviving after 48-h incubation in defined medium without added factors

(Cont) or medium supplemented with 2 ng/ml NGF, 10 ng/ml HGF, or 10

ng/ml anti-HGF expressed as a percentage of the initial count made 3–6 h

after plating. The means and standard errors are shown (n = 9 for each

condition).

Results

HGF promotes the survival of maturing SCG neurons

To investigate if HGF influences the survival of sympathetic

neurons after birth and in the adult, we studied the effects of HGF

on superior cervical ganglion (SCG) neurons in low-density

dissociated cultures established at intervals from postnatal day 1

(P1) to adulthood (P40). For comparison, the neurons were also

cultured with NGF which is known to promote the survival of

sympathetic neurons in the postnatal period. In P1 cultures,

neurons grown with HGF died as rapidly as neurons in control

medium (defined medium with no added growth factors), so that

by 48-h incubation, almost all of the neurons grown under these

conditions had died. In contrast, NGF promoted the survival of the

majority of P1 neurons (Fig. 1A), and survival was neither

enhanced nor reduced by the concomitant presence of HGF (data

not shown). Between P1 and P40, increasing numbers of neurons

survived in control medium. This is due to many of the neurons

acquiring the capacity to survive independently of neurotrophic

factors with age (Orike et al., 2001a). Throughout this period of

postnatal development, between 60% and 80% of the neurons

survived in NGF-supplemented medium. In P10 cultures, signifi-

cantly more neurons survived in HGF-supplemented medium

compared with control medium (40% vs. 20% survival after 48-h

incubation, P b 0.001, t test), and in P20 and P40 cultures, HGF

enhanced neuronal survival as effectively as NGF. Indeed by P40,

the number of neurons surviving with HGF was consistently higher

in the presence of HGF, although this trend did not approach

statistical significance (Figs. 1 and 2). Fig. 2A shows that there was

no additional survival in P40 cultures supplemented with HGF plus

NGF compared with either factor alone at 24, 48, 72, and 96 h data

points, suggesting that HGF and NGF enhance the survival of

essentially the same subset of neurons in the adult. Fig. 2B plots

the dose response of P40 neurons to HGF, showing that a

concentration of 10 ng/ml was maximally effective in promoting

survival. This concentration of HGF was used in all other

experiments.

Because HGF enhances the survival of sympathetic neuroblasts

by an autocrine mechanism, we investigated whether endogenously

produced HGF plays any role in sustaining the survival of postnatal

and adult neurons by studying the effect of function-blocking anti-

HGF antiserum on the survival of these neurons. Figs. 1 and 2A

show that there were no fewer neurons surviving in the presence of

anti-HGF antibody compared with control cultures. To confirm that

the anti-HGF antibody blocks the function of HGF, we set up a

separate series of experiments to investigate if anti-HGF at the

same concentration as that used in the above experiments blocks

the survival-promoting effect of 10 ng/ml HGF in P40 cultures.

After 48-h incubation, the percent survival in cultures supple-

mented with HGF was 88.0% F 9.4% compared with 47.5% F7.3% in cultures supplemented with HGF + anti-HGF, 49.0% F

Fig. 2. (A) Bar chart of the numbers of P40 SCG neurons surviving after 24-, 48-, 72-, and 96-h incubation in defined medium without added factors (Cont) or

medium supplemented with 2 ng/ml NGF, 10 ng/ml HGF, NGF plus HGF, or 10 ng/ml anti-HGF expressed as a percentage of the initial count made 3–6 h after

plating. The means and standard errors are shown (n = 9 for each condition). (B) Graph showing the percentage survival of P40 SCG neurons grown for 48 h

with a range of HGF concentrations. For comparison, the percent survival in control cultures (bar labeled C) is also shown. The means and standard errors are

shown (n = 9 for each condition). (C) Graph showing the level of Met mRNA relative to GAPDH mRNA in total RNA extracted from P1 to P40 SCG. The

mean and standard error of measurements made from three separate assays for each age are shown.

J. Thompson et al. / Mol. Cell. Neurosci. 27 (2004) 441–452 443

5.6% in cultures containing anti-HGF alone, and 42.1% F 4.6% in

control cultures (n = 3 for all data points). Taken together, these

results suggest that secreted HGF does not play a role in sustaining

the survival of postnatal and adult SCG neurons. However, because

intracellular HGF is not accessible to the antibody, we cannot

exclude the possibility that endogenously produced HGF could

bind and activate its receptor intracellularly.

Developmental changes in Met expression

To determine if the age-related effects of HGF on sympathetic

neuron survival are correlated with changes in Met expression, we

used competitive (reverse transcription–polymerase chain reaction)

RT–PCR to quantify the levels of Met mRNA in the SCG from P1

to P60. To compensate for any developmental changes in cell

number and size of the ganglia dissected from different animals,

we also quantified the level of GAPDH mRNA which encodes a

housekeeping enzyme. Fig. 2C shows that Met mRNA was

expressed throughout this period and that there was a gradual

increase in expression after P10, which may indicate acquisition of

the HGF survival response.

HGF enhances the growth of SCG neurites

Previous work has shown that HGF cooperates with NGF to

enhance the growth of embryonic sensory and sympathetic neurites

in culture and that Met signaling is required for the proper growth

of embryonic sensory nerves in vivo (Maina et al., 1997, 1998). To

determine if HGF also affects the growth of postnatal and adult

sympathetic neurites, we used stereological methodology to

quantify the mean total length of the neurite arbors of SCG

neurons grown with and without HGF. Because different subsets of

sympathetic neurons might possess different neurite growth

characteristics independent of the presence of HGF, it was

J. Thompson et al. / Mol. Cell. Neurosci. 27 (2004) 441–452444

important to compare the effect of the presence and absence of

HGF in the same subset of neurons. To achieve this, we

supplemented all cultures with NGF since, as shown in Fig. 1;

no more neurons survive with HGF plus NGF than NGF alone.

The cultures were set up at stages throughout postnatal sympathetic

neuron development, and neurite arbor length was quantified at 24,

48, 72, and 96 hourly intervals after plating. At all ages and all time

points, neurite arbors were longer in cultures containing NGF plus

HGF compared with cultures containing NGF alone. This differ-

ence was statistically significant in two of the four time points in

P1 cultures, all time points in P10 cultures, three time points in P20

cultures, and one time point in P40 cultures (Fig. 3A).

Analysis of neurite growth from P1 SCG neurons grown over a

broad range of HGF concentrations revealed that 10 ng/ml

produced the greatest increase in length of neurite arbors in

Fig. 3. (A) Bar charts of the total length of neurite arbors growing from P1, P10, P2

supplemented with 2 ng/ml NGF, NGF plus 10 ng/ml HGF, or 10 ng/ml anti-H

*Significant difference ( P b 0.05) in neurite length between NGF supplemente

difference ( P b 0.01) in neurite length between NGF supplemented cultures and cu

of P1 SCG neurons grown for 96-h incubation with a range of HGF concentration

Bar chart showing the neurite arbor lengths of Bax-deficient P1 SCG neurons gro

supplemented with either 10 ng/ml HGF or anti-HGF. (D) Bar chart showing the n

growth factors (Cont) and cultures supplemented with either 10 ng/ml HGF or anti

NGF-supplemented medium (Fig. 3B). This was the same level of

HGF that was most effective in promoting neuronal survival at

later ages. These results also demonstrate that HGF is capable of

enhancing neurite growth in the early postnatal period, although it

has no apparent effect on neuronal survival at this stage.

Although the above studies show that HGF cooperates with

NGF to enhance the growth of postnatal sympathetic neurites

beyond that promoted by NGF alone, they do not show if HGF is

capable of promoting neurite outgrowth alone. To address this

question in the immediate postnatal period when most sympathetic

neurons are dependent on NGF for survival, we established

cultures from Bax-deficient embryos. Bax is required for the death

of NGF-deprived sympathetic neurons (Deckwerth et al., 1996), so

by using Bax-deficient neurons, we were able to compare the effect

of the presence and absence of HGF on neurite growth

0, and P40 SCG neurons after 24-, 48-, 72-, and 96-h incubation in medium

GF. The means and standard errors are shown (n = 9 for each condition).

d cultures and cultures supplemented with NGF plus HGF, **Significant

ltures supplemented with NGF plus HGF. (B) Graph of neurite arbor lengths

s. The means and standard errors are shown (n = 9 for each condition). (C)

wn for 72 h in defined medium without growth factors (Cont) and cultures

eurite arbor lengths of P40 SCG neurons grown in defined medium without

-HGF. The means and standard errors are shown (n = 9 for each condition).

J. Thompson et al. / Mol. Cell. Neurosci. 27 (2004) 441–452 445

independently of NGF. Fig. 3C shows that the neurites emerging

from P1 Bax-deficient sympathetic neurons were significantly

longer in the presence of HGF compared with neurons grown with

no factors (P b 0.05, t test). These studies indicate that HGF is

able to promote neurite outgrowth from postnatal sympathetic

neurons independently of NGF.

To explore the possibility that the neurite outgrowth observed

in control cultures of Bax-deficient neonatal SCG neurons may

have been due in part to the secretion of endogenous HGF, we

treated these cultures with function-blocking anti-HGF anti-

bodies. Fig. 3C shows that this treatment did not significantly

reduce neurite outgrowth compared with control cultures,

suggesting that an HGF autocrine loop is unlikely to play a

significant role in promoting neurite growth in this particular

experimental paradigm. Likewise, because a substantial propor-

tion of adult SCG neurons survive independently of added factors

and because anti-HGF does not impair the survival of these

neurons, it was also possible to determine whether endogenously

secreted HGF plays any role promoting neurite growth from adult

neurons. Fig. 3D shows that there was no significant difference

in the total neurite length between control cultures and cultures

treated with anti-HGF. Thus, despite the fact that the majority of

adult mouse SCG neurons coexpress HGF and Met (Yang et al.,

1998), these results suggest that an HGF autocrine loop is

unlikely to play a significant role in promoting neurite growth

from adult sympathetic neurons. Fig. 4 illustrates the typical

appearance of SCG neurons grown under different experimental

conditions at P40.

Fig. 4. Photomicrographs of representative P40 SCG neurons grown with NGF (A

after 96-h incubation, and the neurite arbors were stained with h-tubulin antibod

photographed with a Zeiss confocal microscope. Scale bar = 30 Am.

Role of PI-3 kinase/Akt signaling in mediating HGF survival

To ascertain which signaling pathways mediate the survival

effects of HGF in sympathetic neurons, we investigated the roles to

two pathways that have previously been implicated to various

extents in the survival-promoting effects of NGF in sympathetic

neurons, namely, the PI-3 kinase/Akt and MAP kinase pathways

(Creedon et al., 1996; Crowder and Freeman, 1998; Kuruvilla et

al., 2000; Mazzoni et al., 1999; Rodriguez-Viciana et al., 1994;

Vaillant et al., 1999; Xue et al., 2000). We begun studying the role

of PI-3 kinase/Akt pathway by using Western blotting to ascertain

whether HGF treatment results in phosphorylation of the serine/

threonine kinase Akt, one of the downstream mediators of PI-3

kinase implicated in cell survival. P40 SCG neurons were plated in

defined medium without growth factors for 6 h, then treated for 10

min with either HGF, NGF, or both factors. Western blots of

protein extracts probed with antibodies to phospho-Akt revealed

that both NGF and HGF caused marked and similar increases in the

level of phospho-Akt compared with untreated cultures. The level

of phospho-Akt in cultures treated with NGF plus HGF was similar

to that observed in cultures treated with either factor alone (Fig.

5A). These results indicate that both NGF and HGF promote

phosphorylation of Akt in mature sympathetic neurons and that

these factors in combination do not appear to have an additive

effect on Akt activation.

To investigate the significance of PI-3 kinase signaling in

mediating the HGF survival response in mature sympathetic

neurons, we treated P40 cultures with the PI-3 kinase inhibitor

), HGF (B), NGF + HGF (C), and no factors (D). The cultures were fixed

y followed by FITC-labeled secondary antibody and were visualized and

Fig. 5. (A) Western blot showing Akt phosphorylation in P40 SCG neurons

grown for 6 h in defined medium and stimulated for 10 min with 2 ng/ml

NGF, 10 ng/ml HGF, NGF plus HGF, or nonstimulated (NS). Blots were

probed with anti-phospho-Akt (upper bands) and anti-Akt antibodies (lower

bands) to show similar loading of each lane. (B) Western blot showing Akt

phosphorylation in P40 SCG neurons grown for 6 h in defined medium

alone or medium supplemented with 20 AM LY294002, 20 AM PD98059,

or 1 AM U0126 45 min before stimulation with 10 ng/ml HGF. Blots were

probed with anti-phospho-Akt (upper bands) and anti-Akt antibodies (lower

bands). (C) Bar charts of percent survival of P40 SCG neurons grown for

24 and 48 h in defined medium alone (control) or medium supplemented

with either 2 ng/ml NGF or 10 ng/ml HGF with or without 20 AMLY294002. The means and standard errors are shown (n = 9 for each

condition).

Fig. 6. (A and B) Western blots showing ERK1/2 phosphorylation in P40

SCG neurons grown for 6 h in defined medium alone or medium

supplemented 20 AM LY294002, 20 AM PD98059, or 1 AM U0126 45

min before stimulation with 2 ng/ml NGF or 10 ng/ml HGF. Blots were

probed with antibodies to phospho-ERK1 and ERK2 (upper bands) and

anti-ERK1/2 antibodies (lower bands) to show similar loading of each lane.

(C) Bar charts of percent survival of P40 SCG neurons grown for 24 and 48

h in defined medium alone (control) or medium supplemented with either 2

ng/ml NGF or 10 ng/ml HGF with or without 20 AM PD98059. (D) Bar

charts of percent survival of P40 SCG neurons grown for 24 and 48 h in

defined medium alone (control) or medium supplemented with either 2 ng/

ml NGF or 10 ng/ml HGF with or without 1 AM U0126. The means and

standard errors are shown (n = 9 for each condition).

J. Thompson et al. / Mol. Cell. Neurosci. 27 (2004) 441–452446

LY294002 to see that this would affect the ability of HGF to

enhance neuronal survival. To confirm that LY294002 inhibits Akt

activation by HGF, we examined the level of phospho-Akt in HGF-

treated cultures with and without this inhibitor. Fig. 5B shows that

the phospho-Akt signal in cultures stimulated with HGF in the

presence of LY294002 was similar to that in control cultures,

suggesting that this inhibitor effectively eliminates activation of

Akt by HGF. In the same experiments, the MEK1/MEK2 inhibitors

PD98059 and U0126 did not affect activation of Akt by HGF. Fig.

5C shows that LY294002 was equally effective in blocking the

survival-promoting effects of both NGF and HGF. Whereas the

great majority of neurons survived with either NGF or HGF, the

number of neurons surviving in cultures treated with these factors

plus LY294002 was similar to that in control cultures after 24- and

48-h incubation. These results demonstrate that PI-3 kinase

signaling plays a major role in mediating the survival enhancing

effects of both HGF and NGF.

Role of MAP kinase signaling in mediating HGF survival

To determine if HGF and NGF activate MAP kinase signaling,

we grew P40 SCG neurons in defined medium without growth

factors for 6 h, then treated for 10 min with either HGF or NGF.

Western blots of proteins extracts probed with antibodies to

phospho-ERK1 and phospho-ERK2 revealed that both NGF and

HGF caused marked increases in the level of phospho-ERK1/2

compared with untreated cultures (Figs. 6A and B). These results

indicate that both NGF and HGF promote activation of the ERK1

and ERK2 MAP kinases.

To investigate the significance of MAP kinase signaling in

mediating the HGF survival response in mature sympathetic

neurons, we pretreated P40 cultures with pharmacological inhib-

itors of MEK1 and MEK2, the MAP2 kinases that phosphorylate

ERK1 and ERK2. To confirm that PD98059 and U0126 inhibit

ERK1/2 activation by HGF, we examined the level of phospho-

ERK1 and phospho-ERK2 in cultures treated with these inhibitors.

Figs. 6A and B show that PD98059 and U0126 not only prevented

the increase in ERK1/2 phosphorylation induced by both HGF and

NGF, but also reduced the levels of phospho-ERK1/2 to less than

those observed in nonstimulated neurons. Of the two inhibitors,

U0126 appeared to be the more effective in reducing ERK1/2

phosphorylation. The induction of ERK1/2 phosphorylation by

J. Thompson et al. / Mol. Cell. Neurosci. 27 (2004) 441–452 447

HGF and NGF was not affected by the PI-3 kinase LY294002. Fig.

6C shows that PD98059 significantly reduced, but did not

eliminate, the survival-promoting effects of both NGF and HGF.

After 48-h incubation, there were 15% fewer neurons surviving in

NGF-supplemented cultures and 27% fewer neurons surviving in

HGF-supplemented cultures. Fig. 6D likewise shows that U0126

significantly reduced the survival-promoting effects of both NGF

and HGF somewhat more effectively than PD98059. After 48-h

incubation, there were 30% fewer neurons surviving in NGF-

supplemented cultures and 31% fewer neurons surviving in HGF-

supplemented cultures. These results demonstrate that MAP kinase

signaling contributes to the survival-enhancing effects of HGF and

NGF in mature sympathetic neurons.

HGF-enhanced growth of early postnatal neuron arbors is MAP

kinase-dependent

Investigating the relative roles of PI-3 kinase and MAP kinase

signaling in mediating the neurite growth-promoting effects of

HGF in adult neurons is complicated by the fact that inhibition of

either of these pathways affects neuronal survival. Likewise,

inhibition of PI-3 kinase by LY294002 virtually eliminates the

NGF survival response in P1 SCG neurons (data not shown).

However, at this stage, neither PD98059 nor U0126 affects the

survival of SCG neurons growing with NGF or NGF plus HGF

Fig. 7. (A) Western blot showing ERK1/2 phosphorylation in P1 SCG neurons gro

ng/ml HGF, NGF plus HGF, or nonstimulated (NS). Blots were probed with antibod

(lower bands) to show similar loading of each lane. (B) Western blot showing ERK

alone or medium supplemented with 20 AM PD98059 or 1 AM U0126 45 min befo

were probed with antibodies to phospho-ERK1 and ERK2 (upper bands) and anti-

P1 SCG neurons grown for 24 and 48 h in defined medium alone (control) or med

HGF with or without 20 AM PD98059 or 1 AM U0126. The means and standard

(data not shown). This observation enabled us to investigate the

role of MAP kinase signaling in mediating the neurite growth-

promoting effects of NGF and HGF in postnatal neurons without

neuronal death confounding the results.

To determine if HGF and NGF activate MAP kinase signaling

in P1 SCG neurons, we grew these neurons in defined medium

without growth factors for 3 h, then treated for 10 min with either

HGF, NGF, or both factors together. Western blots of proteins

extracts probed with antibodies to phospho-ERK1 and phospho-

ERK2 revealed that both NGF and HGF increased the levels of

phospho-ERK1/2 compared with nonstimulated cultures (Fig. 7A).

However, in contrast to P40 cultures where both NGF and HGF

caused marked activation of ERK1/2, HGF was less effective in

activating ERK1/2 than NGF at P1. Western analysis was also

carried out to confirm that the MEK1/2 inhibitors PD98059 and

U0126 prevent ERK1/2 phosphorylation by NGF and HGF. Fig.

7B shows that each of these inhibitors virtually eliminated the

phospho-ERK1/2 signal in neurons stimulated with NGF alone or

NGF in combination with HGF following pretreatment with

PD98059 and U0126.

To investigate the significance of ERK1/2 activation in

mediating the neurite growth-promoting actions of NGF and

HGF, we treated P1 SCG neurons grown with NGF alone or

NGF plus HGF with MEK1/2 inhibitors. Fig. 7C shows that

PD98059 and U0126 each significantly reduced, but did not

wn for 3 h in defined medium stimulated for 10 min with 2 ng/ml NGF, 10

ies to phospho-ERK1 and ERK2 (upper bands) and anti-ERK1/2 antibodies

1/2 phosphorylation in P1 SCG neurons grown for 3 h in defined medium

re stimulation with 2 ng/ml NGF or 2 ng/ml NGF plus10 ng/ml HGF. Blots

ERK1/2 antibodies (lower bands). (C) Bar charts of neurite arbor lengths of

ium supplemented with either 2 ng/ml NGF or 2 ng/ml NGF plus 10 ng/ml

errors are shown (n = 9 for each condition).

J. Thompson et al. / Mol. Cell. Neurosci. 27 (2004) 441–452448

eliminate, the neurite growth response of the neurons to NGF alone

and NGF plus HGF. In the absence of inhibitors, neurite arbors

were longer in cultures supplemented with NGF plus HGF

compared with NGF alone, but in cultures treated with inhibitors,

neurite arbors were reduced to the same length in cultures

containing NGF alone and in cultures containing NGF plus HGF.

This suggests that MAP kinase signaling plays a major role in

mediating the neurite growth-enhancing actions of both HGF and

NGF in developing postnatal sympathetic neurons.

Discussion

During embryonic development it has been established that

HGF promotes the survival and differentiation of sympathetic

neuroblasts and enhances the growth of neurites from NGF-

supported, postmitotic sympathetic neurons but does not promote

the survival of these neurons (Maina et al., 1998). To ascertain

whether HGF influences the survival and growth of postnatal and

adult sympathetic neurons, we studied the effects of HGF in

purified cultures of SCG neurons established at stages after birth.

These studies have revealed that, between P10 and P20, HGF

promotes the survival of an increasing proportion of neurons in a

dose-dependent manner. Between P20 and P40, HGF is as

effective as NGF in promoting neuronal survival. The lack of

any additive effect of HGF plus NGF on survival indicates that

these factors promote the survival of the same subset of neurons,

which at P20 and P40 amounts to about a third of the neurons in

the ganglion. The acquisition of the HGF survival response by

sympathetic neurons in the late postnatal period is correlated with a

progressive increase in the levels of transcripts encoding the Met

receptor. Previous work on the roles of HGF in the embryonic

peripheral nervous system has shown that its survival-enhancing

effect on sensory and parasympathetic neurons is only observed in

the presence of another neurotrophic factor (Davey et al., 2000;

Maina et al., 1997). In contrast, in the juvenile and adult

sympathetic nervous system, HGF is capable of promoting

neuronal survival on its own, and this property has facilitated

investigation of the signaling pathways mediating HGF-promoted

survival (below).

Acquisition of the HGF survival response takes place over the

same extended period of postnatal development as the acquisition

of the capacity of many of the neurons to survive independently of

neurotrophic factors. In the present study, up to a third of mouse

SCG neurons are able to survive in serum-free medium without

neurotrophic factors by adulthood. An even larger proportion of

adult rat SCG neurons are able to survive in culture without

neurotrophic factors, and the demonstration that they can do so

when grown as single cells indicates that they require no factors

from other cells to survive (Orike et al., 2001b). These findings

together with the demonstration that the majority of neurons

coexpress HGF and Met in the adult mouse SCG (Yang et al.,

1998) raise the possibility that an HGF autocrine loop may play a

role in sustaining the survival of adult SCG neurons in medium

lacking neurotrophic factors. However, our demonstration that

function-blocking anti-HGF antibody does not affect the survival

of adult SCG neurons suggests that the secretion of HGF from

these neurons does not play a significant role in sustaining their

survival.

In agreement with previous studies of embryonic and neonatal

SCG neurons (Maina et al., 1998; Yang et al., 1998), we have

shown that, throughout the postnatal period and in adult SCG

neurons, HGF enhances neurite growth from neurons incubated in

the presence of NGF. In addition, we have shown that the growth-

enhancing effect of HGF is not dependent on the presence of NGF

because HGF increases neurite length in cultures of Bax-deficient

neonatal SCG neurons surviving without NGF and increases the

length of neurites growing from the subset of adult SCG neurons

that survive independently of NGF. Thus, HGF not only cooperates

with NGF in promoting neurite outgrowth throughout the life of

sympathetic neurons, but also has a direct neurite growth-

promoting effect on these neurons in the postnatal and adult

nervous system.

Although the majority of adult mouse SCG neurons coexpress

HGF and Met (Yang et al., 1998), our results suggest that secreted

HGF acting on its own receptor does not play a significant role in

promoting neurite growth from either neonatal or adult mouse SCG

neurons grown with or without NGF. Function-blocking anti-HGF

antibody that has previously been shown to inhibit the differ-

entiation of mouse sympathetic neuroblasts (Maina et al., 1998)

does not significantly reduce the length of neurites growing from

neurons incubated with NGF at any age studied and does not

reduce the length of neurites from neurons grown without NGF

(either Bax-deficient neonatal neurons or NGF-independent adult

SCG neurons). These findings appear to conflict with the results of

Yang et al. (1998) who showed that anti-HGF antibody reduces

neurite density and neurite elongation in neonatal rat SCG cultures

containing NGF. The conflicting effects of anti-HGF on neurite

growth in these two studies could represent a difference between

the sympathetic neurons of these two species of rodents or could be

related to differences in the experimental paradigms used. For

example, it may be significant that Yang et al. grew SCG neurons

on collagen, whereas we cultured SCG neurons on laminin, which

permits more extensive neurite outgrowth.

The direct survival-promoting effect of HGF on adult SCG

neurons enabled us to investigate which signaling pathways

mediate the antiapoptotic actions of HGF without the complication

of having to supplement the medium with other neurotrophic

factors. We found that pharmacological inhibition of PI-3 kinase

prevented HGF-induced phosphorylation of Akt and eliminated

the HGF survival response, indicating that PI-3 kinase signaling

plays a key role in mediating the antiapoptotic actions of HGF.

Furthermore, pharmacological inhibition of MEK1 and MEK2

prevented HGF-induced phosphorylation of ERK1 and ERK2 and

reduced, but did not eliminate, the HGF survival response,

indicating that MAP kinase signaling also contributes to the

antiapoptotic actions of HGF in adult sympathetic neurons.

Pharmacological inhibition of PI-3 kinase and MAP kinase

signaling in adult SCG had similar effects on the NGF survival

response, suggesting that both HGF and NGF promote the survival

of mature sympathetic neurons by substantially similar intra-

cellular mechanisms. In postnatal rat cerebellar granule cells,

inhibition of PI-3 kinase also prevents the neuroprotective effect of

HGF following treatment with NMDA, quinolinic acid, or low K+-

containing medium (Hossain et al., 2002; Zhang et al., 2000).

However, in contrast with adult sympathetic neurons, inhibition of

MEK1/2 in postnatal cerebellar granule cells does not affect the

neuroprotective effects of HGF. The apparent lack of involvement

of MAP kinase signaling in the neuroprotective effects of HGF on

postnatal cerebellar granule cells is similar to the case for the

survival response of neonatal SCG neurons to NGF in our current

study. Thus, whereas PI-3 kinase signaling plays a key role in

J. Thompson et al. / Mol. Cell. Neurosci. 27 (2004) 441–452 449

mediating the survival effects of NGF and HGF at all ages at

which SCG neurons respond to these factors, MAP kinase

signaling only begins to play a role as the neurons become fully

mature. Studies of neonatal rat sympathetic neurons have reported

that MAP kinase activation plays only a minor role or no role at all

in mediating the survival effects of NGF (Creedon et al., 1996;

Klesse and Parada, 1998; Mazzoni et al., 1999; Virdee and

Tolkovsky, 1996; Xue et al., 2000).

The lack of effect of MEK1/2 inhibition on the survival of

neonatal SCG neurons enabled us to investigate role of MAP

kinase signaling in the neurite growth-promoting effects of NGF

and HGF without effects on neuronal viability complicating

interpretation of the results. Pharmacological inhibition of MEK1

and MEK2 blocked growth factor-induced phosphorylation of

ERK1 and ERK2 and significantly reduced neurite arbors to the

same length in cultures treated with NGF alone and NGF plus

HGF. This suggests that MAP kinase signaling plays a role in

mediating the neurite growth-enhancing actions of both HGF and

NGF in neonatal sympathetic neurons, although some component

of neurite growth is not dependent on this signaling pathway.

Activation of Map kinase by growth factors has previously been

shown to play a role in mediating their neurite growth-promoting

effects on PC12 cells and a variety neurons (Aletsee et al., 2001;

Choung et al., 2002; Klinz et al., 1996; Sjogreen et al., 2000).

Although the key role of PI-3 kinase signaling in mediating the

survival-promoting effects of HGF precluded direct assessment of

the role of this signaling pathway in HGF-induced neurite growth,

there is evidence from studies of mice expressing Met receptors

whose multifunctional docking sites have been replaced with

specific binding motifs for PI3K, Src, or Grb2 that signaling

through PI3K is sufficient to promote outgrowth of specific motor

axons in vivo and to mediate the growth-promoting effects of HGF

on sympathetic neurites in vitro (Maina et al., 2001).

In summary, we have provided evidence for a direct survival-

promoting effect of HGF on juvenile and adult sympathetic

neurons and have shown that HGF is capable of regulating the

growth of sympathetic neurites throughout the postnatal period and

in the adult. The antiapoptotic effect of HGF is dependent on PI-3

kinase signaling and to a lesser extent on MAP kinase signaling,

and the neurite growth-promoting effect depends at least in part on

MAP kinase signaling. These findings clarify the signaling

pathways utilized by HGF to mediate its various effects on

neurons and imply that HGF has an ongoing role in modulating

sympathetic neuron plasticity and function in the mature nervous

system.

Experimental methods

Neuronal culture

Superior cervical ganglia were dissected from P1 to P40 CD1

mice. P1 superior cervical ganglia were trypsinized (0.05% trypsin

in Ca2+/Mg2+-free HBSS) for 15 min at 378C and dissociated by

trituration using fire-polished Pasteur pipette. P20 and older

ganglia were first treated with collagenase (2 mg/ml in Ca2+/

Mg2+ containing HBSS) for 30 min at 48C before trypsinization for

20 min at 378C and trituration. Most of the nonneuronal cells were

removed, and the resulting cell suspensions by differential

sedimentation (Davies, 1986) and the neurons (N90% pure) were

plated in defined, serum-free medium in 35-mm culture dishes

coated with poly-l-ornithine and laminin. Purified recombinant

human HGF (R and D Systems, UK), NGF (Genentech), function-

blocking polyclonal anti-HGF antibodies (R and D Systems) or

pharmacological inhibitors of PI-3 kinase (LY294002, Calbio-

chem, UK), and MEK1/MEK2 (PD98059 and U0126, Calbio-

chem) were added to the cultures at the time of plating. The

concentration at which specific inhibition was observed by each

inhibitor was determined using dose–response curves and the

appropriate controls (data not shown).

To study survival under different experimental conditions,

neurons were plated at low density (100 to 300 neurons per 35-mm

dish), and the number of neurons attached within a 12 � 12 mm

grid in the center of each dish was counted 6 h after plating. The

number of neurons surviving in this grid was then counted at 24

hourly intervals, and the number of surviving neurons in the grid at

these time points was expressed as a percentage of the initial count

taken at 6 h. In each experiment, triplicate cultures were set up for

all conditions.

To study neurite outgrowth, cultures were set up following the

same experimental procedure as before, except that the neurons

were plated at a higher density (400 to 800 neurons per 35-mm

dish). Total neurite length was measured for each experimental

condition using digital stereology (Kinetic Imaging Ltd.), and the

average neurite length per neuron was calculated by dividing this

figure by neuron number per unit area.

Protein extraction and Western blotting/ECL detection

Primary cultures of dissociated P1 and P40 SCG neurons were

grown for 3 to 6 h in culture before being stimulated for 10 min

with HGF or NGF. Pharmacological inhibitors of PI-3 kinase or

MEK1/MEK2 were added 45 min before trophic factor stimula-

tion. Cells were harvested and homogenized in lysis buffer (50 mM

Tris pH 7.5, 1% Triton, 150 mM NaCl, 1 mM EDTA), and total

protein (10 Ag per lane) was resolved in 8% SDS–polyacrylamide

gels and transferred onto polyvinylidene difluoride blotting

membrane (BDH Poole, Dorset, UK) using a Mini Trans-Blot

Electrophoretic Transfer Cell (Biorad, Herts, UK) according to the

manufacturer’s instructions. The membranes were blocked for 1 h

in 5% Milk/Tris-buffered saline containing 0.1% Tween 20

(TBST), incubated with rabbit polyclonal antibodies to either

phospho-ERK1/2 or phospho-Akt (Cell Signalling Technology,

UK) overnight in TBST at 48C and incubated with a horseradish

peroxidase-linked donkey antirabbit IgG secondary antibody

(Amersham Biosciences, UK) for 1 h in 5% milk/TBST. The

membranes were then stripped with 100 mM h-mercaptoethanol,

2% SDS, 62.5 mM Tris-HCl, pH 6.8 for 30 min at 558C and

reprobed with rabbit antibodies to either ERK1/2 or Akt (Cell

Signalling Technology). The membranes were developed using the

ECL Western detection kit (Amersham Pharmacia Biotech,

Buckinghamshire, UK).

Imaging of SCG neurons

Primary cultures of dissociated P40 SCG neurons were grown

for 96 h in culture in the presence of NGF (2 ng/ml), HGF (10 ng/

ml), NGF+HGF, or without added factors (control). After 96 h, the

neurons were fixed in 100% methanol for 10 min, and then washed

three times in 1� phosphate-buffered saline (PBS). Fixed neurons

were permeabilized in 1� PBS 0.1% Triton-X for 30 min and then

blocked for 1 h in 10% bovine serum albumin (BSA)/PBS (Sigma).

J. Thompson et al. / Mol. Cell. Neurosci. 27 (2004) 441–452450

Anti h-tubulin II monoclonal antibody (1:1000) (Promega) in 10%

BSA/PBS was added to the fixed neurons and left to incubate

overnight at 48C. The following day primary antibody was

removed, and neurons were washed three times in 1� PBS.

FITC-labeled Ig antimouse secondary antibody (1: 2000) (Sigma)

was added and incubated for 1 h. Neurons were washed three times

in 1� PBS and photomicrographs taken using the confocal

microscope.

Measurement of Met mRNA levels

A quantitative competitive reverse transcription–polymerase

chain reaction (RT–PCR) technique (Wyatt and Davies, 1993) was

used to measure the levels Met mRNA in total RNA extracted from

P1 to P60 SCG. To compare the relative levels of the receptor in

different samples, the cRNA competitor template for the ubiq-

uitous, constitutively expressed GAPDH was also included in the

reverse transcription reactions. Total RNA was extracted (Chomc-

zynski and Sacchi, 1987) treated with DNase, purified using an

RNAID kit (BIO101), and recovered in 40 Al of DEPC-treated

H2O. The RNA was reverse-transcribed for 1 h at 378C with

MMuLV-reverse transcriptase, RNaseH (Biogene Ltd., UK) in a

40-Al reaction containing the manufacturer’s buffer supplemented

with 0.5 mM dNTPs, and 10 AM random hexanucleotides. A 5-Alaliquot of each reverse transcription reaction was then amplified in

a 50-Al PCR reaction containing 1� PC2 buffer (Helena

Biosciences), 0.1 mM dNTPs, 2 U of Taq Supreme (Helena

Biosciences), 40 ng of each primer. The forward and reverse assay

primers for Met cDNA were: 5V-CCAGTCCTATATTGATGTC-3Vand 5V-TTCGAAGGCATGTATGTAC-3V. Met cDNA was amplified

by cycling at 958C for 1 min, followed by 1 min at 488C, followed1 min at 688C, for 37 cycles. The reaction was then completed with

a 10-min extension at 688C. For GAPDH cDNA, cycling

conditions were the same except only 29 cycles were carried out

and the annealing temperature was 558C. The RT–PCR products

were run on 8% nondenaturing polyacrylamide gels and visualized

by staining with syber gold (Molecular Probes, Leiden, Nether-

lands). The intensities of the RT–PCR products of the native

mRNA and competitor cRNA were calculated using Phoretix 1D

Quantifier v4.01 (Biogene Ltd.). The amount of Met or GAPDH

mRNAs in each total RNA sample was calculated from these

values.

Acknowledgments

This work was supported by a grant from the Wellcome Trust.

Mary Tolcos was supported by a Howard Florey Research

Fellowship. Xavier Dolcet was supported by a Spanish Ministry

of Education Fellowship.

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